Glycoproteins mediate many essential functions in humans and other mammals, including signalling, cell-to-cell communication and molecular recognition and association. Antibodies or immunoglobulins are glycoproteins that play a central role in the humoral immune response and that are used increasingly as therapeutics. Antigen-specific recognition by antibodies results in the formation of immune complexes that may activate multiple effector mechanisms.
There are five major classes of immunoglobulins (Igs): IgA, IgD, IgE, IgG and IgM. Several of these may further be divided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3 and IgG4. Papain digestion of antibodies produces two identical antigen binding fragments called Fab fragments and a residual Fc fragment. In human IgG molecules, the Fc region is generated by papain cleavage N-terminal to Cys 226. The Fc region is central to the effector function of the antibodies and interaction with various molecules, such as Fcγ receptors (FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and FcγRIIIb), rheumatoid factor (RF), Protein G and A, complement factors (C3b, C1q) and lectin receptors (MBL, MR, DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin)). The interaction of antibodies and antibody-antigen complexes with cells of the immune system mediates a variety of responses, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). In order to be useful in therapy, an antibody, or a fragment thereof, should therefore have suitable effector functions.
The Fc domain sequence of IgG comprises a single site for N-linked glycosylation within its CH2 domain at an asparagine residue 297 (Asn297) numbered according to the EU index (Kabat et al., Sequences of proteins of immunological interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242). Typically the oligosaccharide structures attached to the Fc domain comprise biantennary chains with varying galactosylation.
It is known that the oligosaccharide structure attached to the Fc domain influences the binding of IgG to Fc receptors and other molecules that interact with the antibody molecule, such as C1q (Raju 2008, Curr Opin Immunol 20, 471-478). Thus variations in the oligosaccharide structure (i.e. different glycoforms) of the Fc domain influence ADCC and CDC activity. Subsequently, modification of said oligosaccharide structure may affect the therapeutic activity of an antibody or a fragment thereof. The ability to produce glycoproteins and compositions comprising thereof that are enriched for particular oligosaccharide structures is highly desirable.